RESUMEN
Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.
Asunto(s)
Frío , Citoprotección/efectos de los fármacos , Células Endoteliales/citología , Oxígeno/efectos adversos , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Aorta/citología , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Modelos Moleculares , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores de Muerte Celular/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.
Asunto(s)
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Piridinas/farmacología , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación hacia Abajo/efectos de los fármacos , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/patología , Roscovitina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the present study, to further elucidate the molecular events that control neural crest cell migration, we have analyzed in vitro the adhesive and locomotory response of avian trunk neural crest cells to laminin-1 and searched for the integrin receptors involved in this process. Adhesion of crest cells on laminin-1 was comparable to that found on fibronectin or vitronectin. By contrast, migration was significantly greater on laminin-1 than on the other substrate molecules. Interaction of crest cells with laminin-1 involved two major cell-binding domains situated in different portions of the molecule, namely the E1' and E8 fragments, which elicited different cellular responses. Cells were poorly spread on the E1' fragment whereas, on E8, they were extremely flattened and cohesive. Either fragment supported cell locomotion, albeit not as efficiently as laminin-1. Immunoprecipitation and immunocytochemistry analyses revealed that crest cells expressed the alpha1beta1, alpha3beta1, alpha6beta1 and alpha vbeta3 integrins, as well as beta8 integrins, as presumptive laminin-1 receptors, but not alpha6beta4 and alpha2beta1. Immunofluorescence labeling of cultured cells showed that the alpha1, alpha v, beta1 and beta3 subunits were diffuse on the cell surface and in focal contacts. In contrast, alpha3 and beta8 were diffuse, while alpha6 was mostly intracytoplasmic and, secondarily, in focal contacts. Inhibition assays of cell adhesion and migration with function-perturbing antibodies demonstrated that alpha1beta1 played a predominant role in both adhesion and migration on laminin-1 and interacted with either binding sites in the E1' and E8 fragments. Alpha vbeta3 was also implicated in neural crest cell migration. In contrast, alpha3beta1, alpha6beta1 and the beta8 integrins appeared to play only subsidiary roles in cell adhesion and migration. Finally, the ability of neural crest cells to interact with laminin-1 was found to increase with time in culture, possibly in correlation with changes in alpha3 distribution on the cell surface. In conclusion, our study indicates that (1) the preferential migration of neural crest cells along basal laminae can be accounted for by the ability of laminin-1 to promote migration with great efficiency; (2) interaction with laminin-1 involves two major cell binding domains that are both recognized by the alpha1beta1 integrin; (3) alpha1beta1 integrin can elicit different cellular responses depending on the laminin-1 domains with which it interacts; and (4) changes in the repertoire of integrins expressed by neural crest cells are consistent with the modulations of cell-substratum adhesion occurring throughout migration.